Tracking bacterial pathogens with genetically-encoded reporters

AbstractDuring the infectious process, bacterial pathogens are subject to changes in environmental conditions such as nutrient availability, immune response challenges, bacterial density and physical contacts with targeted host cells. These conditions occur in the colonized organs, in diverse regions within infected tissues or even at the subcellular level for intracellular pathogens. Integration of environmental cues leads to measurable biological responses in the bacterium required for adaptation. Recent progress in technology enabled the study of bacterial adaptation in situ using genetically encoded reporters that allow single cell analysis or whole body imaging based on fluorescent proteins, alternative fluorescent assays or luciferases. This review presents a historical perspective and technical details on the methods used to develop transcriptional reporters, protein–protein interaction assays and secretion detection assays to study pathogenic bacteria adaptation in situ. Finally, studies published in the last 5years on gram positive and gram negative bacterial adaptation to the host during infection are discussed. However, the methods described here could easily be extended to study complex microbial communities within host tissue and in the environment

Mapping of Shigella flexneri’s tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs

International audienceShigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo